As a result of recent studies, the possibility of practical use of human pluripotent stem cells such as human ES cells (hESC) or human iPS cells (hiPSC) has been increased in the field of regenerative medicine. These human pluripotent stem cells have an infinite proliferative ability and an ability to differentiate into various cells, and thus, the cells are expected as a therapeutic means for treating intractable disease, lifestyle-related disease and the like. It has been demonstrated that human pluripotent stem cells can be induced to differentiate in vitro into a variety of cells such as nerve cells, cardiomyocytes, blood cells, or retinal cells.
Feeder cells act to supply a growth factor for the maintenance culture of human pluripotent stem cells to stein cells. Thus, human pluripotent stem cells such as hESC or hiPSC have been conventionally cultured mainly on a layer of mouse-derived feeder cells (MEF: mouse embryonic fibroblasts). Activity of performing the maintenance culture of human pluripotent stein cells has also been reported for various human cells (Non Patent Literatures 1 to 4). However, the method of using mouse-derived feeder cells has been problematic in terms of safety to living bodies because the feeder cells are mixed into human pluripotent stem cells. In addition, a culture method of using human-derived feeder cells has also been problematic in terms of time-consuming preparation of feeder cells upon culture.
As methods for culturing human pluripotent stem cells without using feeder cells such as MEF, a method of previously conditioning a medium with MEF (MEF-CM) and a method of chemically immobilizing MEF on a medium have been known (Non Patent Literature 5). Moreover, a method of using, as living feeder cells, various human-derived cells such as fibroblasts, placental cells, bone marrow cells or endometrial cells, without using heterologous cells, has also been reported (Non Patent Literature 6). Furthermore, in order to culture human pluripotent stem cells, a medium containing bovine serum, KNOCKOUT™ SR (Knockout Serum Replacement: an additive that can be used, instead of serum, to culture ES/iPS cells), and the like has been used. However, such a medium often contains a protein extracted from bovine serum, and thus, it has been concerned about infectious diseases such as bovine spongiform encephalopathy (BSE) or cell contamination caused by virus. There is a case where human-derived serum is used, but since such human-derived serum has restrictions and limitations on use, it is not suitable for practical use. Further, it has been known that human pluripotent stem cells can be cultured in the absence of feeder cells by using a medium for human pluripotent stem cells containing such serum or a serum replacement, which has been conditioned with MEF. However, it has been difficult to identify a factor useful for the growth of human pluripotent stem cells, which is secreted from MEF, in a medium containing a large amount of serum-derived protein. In order to identify such a factor, a receptor protein that changes specifically in human ES cells cultured in MEF-CM has also been analyzed (Non Patent Literature 7).
The development of a chemically defined medium for carrying out a culture without using MEF has also been promoted, and the problem regarding contamination with bovine serum-derived components has been almost avoided (Non Patent Literatures 8 and 9). Using an MEF secretion, the analysis of functional proteins has been carried out (Non Patent Literature 10). Also, it has been reported that embryonic stem cells can be cultured without using feeder cells by addition of vitronectin and IGF1 chimeric protein (Non Patent Literature 11). As commercially available IGF-containing media, mTeSR1 (registered trademark) manufactured by STEM CELL Technologies, STEMPRO (registered trademark) manufactured by Life Technologies, and the like have been known. However, these media have been problematic in that human pluripotent stem cells cannot be stably cultured therein, and in that they exhibit poor proliferative ability therein.
On the other hand, Patent Literature 1 describes a method for producing a composition comprising a Vitamin K-dependent protein of interest with a substantially lower amount of at least one protein contaminant expressed endogenous by the host cell in the absence of modification, the method comprising the steps of a) producing a host cell according to the invention; and b) growing the host cell in a growth medium and harvesting the growth medium comprising the Vitamin K-dependent protein of interest. Patent Literature 1 also describes GAS-6 as an example of the vitamin K-dependent protein (paragraphs 0008 and 0035). Patent Literature 2 describes that cells are cultured in a medium containing 10% FCS and GAS6 when a scratch assay is carried out (paragraph 0059). In addition, Patent Literature 3 describes that gas6 is added as a neuron growth enhancer to a medium (paragraph 0235). However, there have been no reports regarding addition of GAS6 to a serum-free medium.
Moreover, GAS6 has been clarified to be a ligand for receptor tyrosine kinase (Non Patent Literature 12). The receptor tyrosine kinase includes EGFR, ERBB2, ERBB3, ERBB4, INSR, IGF-1R, IRR, PDGFRα, PDGFRβ, CSF-1R, KIT/SCFR, FLK2/FLT3, VEGRF 1 to 3, FGFR1 to 4, CCK4, TRKA, TRKB, TRKC, MET, RON, EPHA 1 to 8, EPHB 1 to 6, AXL, MER, TYRO3, TIE, TEK, RYK, DDR1, DDR2, RET, ROS, LTK, ALK, ROR1, ROR2, MUSK, AATYK, AATYK2, AATYK3, RTK106, and the like. It has been reported that GAS6 functions as a ligand for receptor tyrosine kinase belonging to the TAM family (TYRO3, AXL, and MER) (Non Patent Literature 13).